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High Content Analysis - Day 3

 

Friday, January 14

8:00 am Morning Coffee

 

Novel Biological Models for HCA

8:30-8:35 Chairperson’s Opening Remarks

Steven Haney, Ph.D., Associate Fellow, Biological Profiling, Pfizer Research Technology Center

8:35-9:00 HCA of Complex Cell Models for Single Agent and Combination Studies of Drug Efficacy

Bev Isherwood, Ph.D., Associate Principal Scientist, Research & Development, Advanced Science and Technology Laboratory, AstraZeneca

Incorporation of complex biological models into drug discovery programs promises to deliver screens with improved physiological relevance and better early predictability of compound effects in the clinic. We describe the development of co-culture, 3D and kinetic models of biological processes in combination with the latest developments in high-content analysis and laboratory automation to profile compound mode of action. We demonstrate that complex biological systems are translatable to medium/high throughput techniques allowing larger compound sets to be profiled faster, more consistently and in combination.

9:00-9:25 Application of 3-D Tumor Cell Culture Model to Compound Screening

Miroslav Cik, Ph.D., Research Fellow, Assay Development and Target Validation, Johnson & Johnson Pharmaceutical R&D

Adherent cell growth in a 2D model does not represent the complex cellular environment that is present in tissues and organs. To mimic more closely the in vivo situation and tumor microenvironment, we need to use a 3-dimensional (3D) cell culture model where tumor cells are co-cultured with primary human fibroblasts. This model better simulates the tumor microenvironment and signaling pathways that are present in vivo. We will present our experience with setting up a larger scale 3D culture and high-content assay. Screening results of a compound library collection of small molecules will also be presented.

9:25-9:50 C. Elegans as a Tool for Drug Discovery

Stephen C. Pak, Ph.D., Research Assistant Professor, University of Pittsburgh School of Medicine

9:50-10:15 Understanding the Genetics of Type II Diabetes through Cellular Tissue Models: Novel Genes Involved in Hepatic Glucose Regulation as an Example

Steven Haney, Ph.D., Associate Fellow, Biological Profiling, Pfizer Research Technology Center

Complex diseases such as Type II Diabetes have been a challenge to understand due to the multifactorial nature of disease incidence and progression, as well the interplay between organs that contribute to serum glucose regulation. While genome-wide association studies have been valuable in defining a relatively few number of loci that harbor mutations which contribute to disease incidence, the number of candidate genes is still relatively large and many are uncharacterized. Cellular models that faithfully recapitulate the in vivo cellular biology are essential to investigating candidate gene function. We have been successful in assigning the function of novel genes to hepatic glucose regulation through a combination of a human primary hepatocyte cell culture system that carefully represents the natural biology of liver hepatocytes and quantitative cytology to measure glucose regulation and cellular signaling following perturbations by siRNAs to candidate genes.

10:15-10:50 Coffee Break

10:50-11:15 Potential Biomarker Identification via Automated Analysis of Subcellular Patterns in Tissue Microarrays

Robert F. Murphy, Ph.D., Professor, Computational Biology and Biological Sciences, Biomedical Engineering, and Machine Learning; Director, Ray and Stephanie Lane Center for Computational Biology, Carnegie Mellon University

Automated analysis of subcellular location is critical for drug and diagnostic development. We have extended previous pattern analysis work for cultured cells to proteome-scale image collections obtained using tissue microarrays. The task is quite challenging because dense packing of cells complicates segmentation. We have therefore developed a range of features that do not require segmentation and showed that they can be used to recognize eleven major subcellular patterns with over 95% accuracy. We have also used these methods to identify a number of potential biomarkers associated with cancer from a number of different tissues. 

11:15-11:40 High-Content Imaging of Differentiation and Epigenetics of Human Pluripotent Stem Cells

Paul Sammak, Ph.D., Research Associate Professor, Department of Cell Biology and Physiology, The University of Pittsburgh

High-Content Screening of transcription factors that are characteristic of pluripotent or differentiated stem cells was used to develop new protocols for early development of trophectoderm, placental precursor cells, from human embryonic stem cells (hESC). Morphological transformation of hESC colonies to trophectoderm was measured over time non-invasively by texture analysis, demonstrating the uniformity and efficiency of the differentiation process. Further, the histone deacetylase (HDAC) inhibitor TSA slows BMP4 induced differentiation, demonstrating that chromatin acetylation changes are required during differentiation. Further, the ability to form trophectoderm is characteristic of totipotency and can be used to distinguish these more primitive pluripotent cells.

11:40-12:05 pm Phenotypic Profiling of Primary Human Macrophages to Drugs and RNAi during Mycobacteria Phagocytosis

Marc Bickle, Ph.D., Head, HT Technology Development Studio, Max Planck Institute of Molecular Cell Biology & Genetics

We have developed a high-content assay in primary human macrophages that measures intracellular survival of mycobacteria using high-resolution imaging on the OPERA from Perkin Elmer. We monitor the intracellular survival of M. bovis (BCG strain) and simultaneously measure cellular response of the primary human macrophages. Using this approach, we have discovered several compounds affecting either the host or the bacterium. We are also developing an RNAi approach to discover host genes involved in arresting the maturation of phagosomes. To transfect the primary human macrophages we are developing high-throughput electroporation protocols on the Cellaxess HT system of Cellectricon.

12:05-12:30 Facing the Challenges of High-Content Screening in Leishmania Amastigotes-Hosting Primary Macrophages

Nathalie Aulner, Ph.D., Research Engineer, PFID - Imagopole, Institut Pasteur

Leishmaniases are caused by the protozoan parasites Leishmania and are potentially fatal diseases. In areas where these human diseases are endemic, most often the Leishmania are hosted by wild rodents and blood-feeding sand flies. Leishmania alternate between the insect flagellated promastigote stage and the amastigote stage that proliferates within a parasitophorous vacuole/PV of rodent macrophages. Leishdrug, a European FP7 funded interdisciplinary project, aims to exploit Leishmania amastigotes-specific pathways; it uses a highly multidisciplinary strategy for leismanicidal drug development. An established bench-top visual phenotype-based assay utilizing model rodent-namely laboratory mouse-bone marrow-derived macrophages and DsRed2 transgenic L. amazonensis amastigotes purified from nude mouse footpad lesions has been adapted and up-scaled to the exigence of a robust high-content screening methodology. The high-content strategy relies on the detection and counting of fluorescent amastigotes, PVs and viable macrophages as read-outs and allows the simultaneous assessment of selective leishmanicidal activity and host cell toxicity under physiological conditions. The challenge of working with physiologically relevant primary macrophages as Leishmania-hosting cells and the clinically relevant amastigote stage for assay miniaturization, the statistics and sampling approaches as well as preliminary results from pilot screens will be described.

12:30 Close of Conference



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